1. Field of the Invention
This invention relates to an article having a non-thrombogenic surface and a process for making the article. More particularly, this invention relates to an article formed by (i) coating a polymeric substrate with a crosslinked chemical combination of a polymer having at least two amino substituted side chains, a crosslinking agent containing at least two crosslinking functional groups which react with amino groups on the polymer, and a linking agent containing a first functional group which reacts with a third functional group of the crosslinking agent, and (ii) contacting the coating on the substrate with an antithrombogenic agent, such as heparin, which covalently bonds to a second functional group of the linking agent.
2. Description of the Related Art
It is well known that when blood comes into contact with a surface other than the natural wall of a blood vessel, the activation of certain circulating substances results in the coagulation of the blood. If thrombi are formed on portions of the surface which contact blood flow, there is a risk that the thrombi will be released and cause serious blood circulation disturbances called thrombosis. As a result, extensive investigations have been undertaken over many years to find materials having a reduced tendency to form thrombosis. This area of research has become increasingly important with the development of various objects and articles which can be in contact with blood, such as artificial organs, vascular grafts, probes, cannulas, catheters and the like.
Synthetic polymeric materials have come to the fore as preferred materials for such articles. However, these polymeric materials have the major drawback of being thrombogenic. Accordingly, numerous procedures for rendering a polymeric surface non-thrombogenic have been proposed. (As used herein, xe2x80x9cnon-thrombogenicxe2x80x9d and xe2x80x9cantithrombogenicxe2x80x9d refer to any material which inhibits thrombus formation on a surface.) One well known approach for counteracting thrombogenicity of a polymeric surface has been the use of antithrombogenic agents or anticoagulants such as heparin. Heparin is a highly sulfated dextrorotatory mucopolysaccharide composed of D-glucosamine and D-glucuronic acid residues, and is known to prolong the clotting time of blood.
Various general methods for the attachment of heparin to otherwise thrombogenic polymeric surfaces are known. In one general method, heparin is ionically bound to a surface. Heparin is an anionic compound which easily forms ion complexes with cationic compounds. As a result, it has been proposed to attach a cationic substance to a surface and thereafter ionically bind heparin to the cationic substance. For example, U.S. Pat. No. 3,617,344 discloses a method in which a polymeric surface is chemically modified to include a chloromethyl group, the chloromethyl group is aminated to provide a quaternary ammonium halide, and the halide is reacted with sodium heparin to ionically bond heparin to the surface. One disadvantage with ionically bound systems is that the heparin can leach off the surface when contacted with blood or other fluids.
Because of the leachability of ionically bound heparin, another general method for the attachment of heparin to otherwise thrombogenic polymeric surfaces has been developed wherein heparin is covalently bound to a surface. Immobilization of heparin to artificial blood-contacting materials through covalent bonding has proven to be a successful approach for achieving a non-thrombogenic surface suitable for use in medical applications. Previous efforts to covalently immobilize heparin include: (1) the formation of an amide linkage derived from the xe2x80x94CO2H of heparin and a polymer carrying an NH2-side-chain by coupling with a water-soluble carbodiimide (see, for example, U.S. Pat. No. 4,521,564); (2) the formation of an ether group by reaction of the xe2x80x94OH group of the sugar ring with an epoxidized support; and (3) the linking of heparin at its reducing end to an xe2x80x94NH2 containing solid matrix by reductive amination (see U.S. Pat. No. 4,810,784). According to the last approach, a polyethylene substrate was modified by (i) brief treatment with KMnO4 in concentrated sulfuric acid to generate anionic (xe2x80x94CO2H/SO3H) sites, (ii) incubation with 0.01%. polyethylenimine, and (iii) coupling of the resulting NH2-rich surface with heparin by reductive amination (NaBH3CN in buffer at pH 3.5). Apart from the advantage of its long-term stability (reportedly up to several months), the heparin incorporated this way (the so-called xe2x80x9cend-point attachmentxe2x80x9d) mimics its natural configuration, allowing maximal retention of its antithrombogenic properties.
It has been established further, that the end-point attachment technique can be successfully extended to polymeric carriers bearing surface hydrazide groups (See D. J. O""Shannessy and M. Milcheck, Anal. Biochem. 191, 1-8, 1990). Hydrazide is much more active over xe2x80x94NH2 as a nucleophile in reaction with aldehydes (including all reducing sugars), while possessing lower basicity in comparison to amines (pK for hydrazides: xcx9c3, for primary amines: xcx9c7). Notable advantages of using a hydrazide matrix for immobilization of reducing sugars, including heparin, are: (1) a faster reaction (about 30-fold for simple saccharides) than using the xe2x80x94NH2 supports (See Y. Ito, Y. Yamasaki, N. Seno, and I. Matsumoto, J. Biochem. Tokyo, 99, 1267-1272, 1986); (2) the reaction of hydrazide with xe2x80x94CHO is an irreversible process and therefore, the need for further stabilization by NaBH3CN reduction can be partly avoided or totally eliminated; and (3) unlike primary amines, hydrazides remain unprotonated at slightly acidic pH levels (as low as 3-4.7). Reaction under these conditions will help to minimize the possible by-products originating from the xe2x80x94NH2 groups in the substrate and coating materials.
A number of solid supports (mostly in the form of polysaccharide beads) containing hydrazide groups are presently commercially available for use as adsorbents in affinity chromatography. These hydrazide supports may be prepared by: (1) diimide coupling of polymeric amines with p-hydrazinobenzoic acid; (2) direct condensation of an epoxy-containing polymer with a dihydrazide like adipic dihydrazide; and (3) coupling of polymeric active esters with hydrazine. The preparation of hydrazide supports and their application in affinity chromatography of oligosaccharides, polysaccharides, glycoproteins, and enzymes carrying sugar units is described in a number of patents (See, for example, U.S. Pat. Nos. 4,217,338, 4,419,444, 4,874,813, 4,948,836, 5,104,931, 5,316,912, and Japanese Patent Publication No. 59015401.) Immobilization is carried out by reaction of the hydrazide reagent with the reducing terminus of the target molecule. Alternatively, the hydrazide coupling is preceded by a periodate-oxidation (to split the vicinal diols of sugar unit and provide newly generated xe2x80x94CHO groups) and finally completed by NaBH3CN reduction.
The preparation of hydrazide matrices has been reported in the technical literature. For example, (1) the preparation of modified polysaccharide matrices (cellulose, Sephadex, and Sepharose) through NalO4-oxidation and subsequent reaction with adipic dihydrazide is described by E. Junowicz, and S. E. Charm at Biochim. Biophys. Acta 428, 157-165, 1976; (2) the preparation of polyacrylhydrazide-agarose by periodate oxidation of agarose followed by reaction with polyacrylhydrazide is described by T. Miron and M. Wilchek at J. Chromatogr. 215, 55-63, 1981; (3) the preparation of polyacrylamide-polyhydrazides from the corresponding N-hydroxysuccinimide-ester and hydrazine and use in the analysis of glycoproteins is described by U. Heimgartner, B. Kozulic, and K. Mosbach at Anal Biochem. 181, 182-189, 1989; and (4) the preparation of hydrazide-derivatized Eupergit C beads from Eupergit C [a poly(methyl methacrylamide) bearing epoxide group] and adipic dihydrazide is described by G. Fleminger, E. Hadas, T. Wolf, and B. Solomon at Applied Biochem. Biotechnol. 23, 123-137, 1990.
Other techniques for the immobilization of heparin and related sulfated sugars on various substrates are described in the technical literature. For example, (1) heparin and dermatin sulfate immobilized on hydrazide-Toyopearl for isolation of lectin is described by H. Hitagaki, H. Motsumoto, H. Sasaki, and N. Seno at J. Biochem. (Tokyo), 98, 385-393, 1985; (2) partially periodate-oxidized heparin and others on immobilized adipic dihydrazide-agarose for studying glycoprotein-heparin interactions is described by M. Del Rosso et al. at Biochem. J. 199, 699-704, 1981; and (3) heparin immobilized on adipic dihydrazide modified poly(methyl vinyl ether-alt-maleic anhydride) is described by A. Satoh, K. Kojima, T. Koyama, H. Ogawa, and I. Matsumoto, at Anal. Biochem. 260, 96-102, 1998 and by K. Isosaki, N. Seno, I. Matsumoto, T. Koyama, and S. Moriguchi, at J. Chromatogr. 597, 123-128, 1992 for use in ELISA.
Even though various techniques are known for attaching heparin and other antithrombogenic agents to a substrate, there is still a need for an improved antithrombogenic polymer coating that may be easily applied to a substrate to provide a material which has excellent biological and chemical stability towards blood and which retains its antithrombogenic properties in a permanent and non-leachable fashion when in contact with blood for prolonged periods.
The foregoing needs are met by an article having a non-thrombogenic surface according to the present invention and by a process for rendering the surface of a substrate non-thrombogenic according to the present invention. An article according to the invention comprises a substrate, a coating disposed on at least a portion of the substrate, and an antithrombogenic agent covalently bonded to the coating. The coating comprises a crosslinked chemical combination of (i) a polymer having side chains along a backbone forming the polymer, at least two of the side chains containing an amino group, (ii) a crosslinking agent containing at least two functional groups capable of reacting with the amino groups, and (iii) a linking agent containing a first functional group and a second functional group, the first functional group capable of reacting with the crosslinking agent""s functional groups. The antithrombogenic agent is covalently bonded to the second functional group of the linking agent.
The substrate of an article according to the invention may comprise any polymeric material conventionally used to fabricate articles commonly used in contact with blood. The substrate serves as a support for the coating and the antithrombogenic agent.
The polymer used in the coating comprises a polymer having side chains along a backbone forming the polymer wherein at least two of the side chains contain an amino group (xe2x80x94NRH, xe2x80x94NH2, xe2x80x94NRH2+, xe2x80x94NH3+). In one example embodiment, the polymer is a polyamide having amino substituted alkyl chains on one side of the polymer backbone.
The crosslinking agent used in the coating contains at least two functional groups capable of reacting with the amino groups of the polymer used in the coating. In one example of the crosslinking agent used in the coating, the crosslinking agent is selected from the group consisting of phosphines having the general formula (A)3P, wherein A is hydroxyalkyl. One more specific example of the crosslinking agent used in the coating is tris(hydroxymethyl)phosphine.
The linking agent used in the coating contains a first functional group and a second functional group wherein the first functional group is capable of reacting with a third functional group of the crosslinking agent. In one example of the linking agent used in the coating, the linking agent is a polyhydrazide, that is, the linking agent includes at least two functional groups having the formula xe2x80x94CONHNH2. One specific example of the linking agent used in the coating is adipic dihydrazide.
The antithrombogenic agent used in an article according to the invention may be any material which inhibits thrombus formation on its surface, such as by reducing platelet aggregation, dissolving fibrin, enhancing passivating protein deposition, or inhibiting one or more steps within the coagulation cascade. In one example of the antithrombogenic agent, the antithrombogenic agent is selected from heparin, prostaglandins, urokinase, streptokinase, sulfated polysaccharide, albumin and mixtures thereof. One more specific example of the antithrombogenic agent is heparin.
The article having a non-thrombogenic surface may be produced by a process according to the invention in which a polymer having at least two amino substituted side chains is mixed with a crosslinking agent and a linking agent to produce a polymer solution. The crosslinking agent contains at least two crosslinking functional groups which react and combine with amino groups on the polymer, and a third functional group. The linking agent contains a first functional group which reacts and combines with the third functional group of the crosslinking agent, and a second functional group. The polymer solution is coated on at least a portion of a substrate to produce a crosslinked polymer coating on the substrate. At least a portion of the crosslinked polymer coating on the substrate is then contacted with an antithrombogenic agent such that the antithrombogenic agent covalently bonds to the second functional group of the linking agent.
In an example embodiment of the invention, the versatile chemical methodology of the invention allows the attachment of heparin through covalent linkage to a two-dimensional polymer carrier that is deposited on a polymeric substrate (e.g., polydimethylsiloxane, polyurethane, and polypropylene). The two-dimensional polymers have a backbone of repeating xcex2-amino acid units with long aliphatic side-chain and free NHxe2x80x94 and NH2xe2x80x94 substituents and are synthesized by condensation of 2(5H)-furanone, or maleic acid derivatives (such as anhydride, esters, and so on) with a long-chain amine (e.g., tetradecylamine) and a polyamine (e.g., pentaethylenehexamine). Coupling of the two-dimensional polymer with tris(hydroxymethyl)phosphine (the crosslinking agent) and adipic dihydrazide (or other di-, tri-, and polyhydrazide linking agents having at least two xe2x80x94CONHNH2 groups) results in the formation of a triblock polymer with pendant hydrazide groups. The coupling solution is used (without isolation) directly for the preparation of an intermediary reactive coating. The latter is then allowed to react with heparin or heparin/sodium cyanoborohydride in aqueous medium to produce a covalently bonded antithrombogenic surface with remarkably enhanced heparin content (greater than or equal to 10 micrograms/cm2) and improved operational stability. Direct heparinization with sodium heparin forms a hydrazone, while heparinization by reductive amination forms a reduced hydrazone.
It is an advantage of the present invention to provide an improved antithrombogenic polymer coating that may be easily applied to a substrate to provide a material which has excellent biological and chemical stability towards blood and which retains its antithrombogenic properties in a permanent and non-leachable fashion when in contact with blood for prolonged periods.
It is another advantage of the invention to provide a process for the preparation of a two-dimensional-polymer surface containing pendant reactive hydrazide groups that may be further attached to an antithrombogenic agent such as heparin.
It is yet another advantage to provide a process for the preparation of a two-dimensional-polymer surface containing pendant reactive hydrazide groups that is easily realized by coupling the polymer with a crosslinking agent in the presence of a linking agent (i.e., spacer-arm) containing at least two hydrazide groups wherein the intermediary layer from the copolymer thus obtained demonstrates excellent ability to form a stable linkage with the substrate while leaving its pendant hydrazide groups for further attachment to an antithrombogenic agent such as heparin.
It is still another advantage to provide a process for immobilizing heparin to a reactive coating surface and to provide a process for multiplication of reactive sites through activation of NH2xe2x80x94, NHxe2x80x94, or hydrazide groups on the coating surface followed by coupling with a trihydrazide or polyhydrazide.
These and other features, aspects, and advantages of the present invention will become better understood upon consideration of the following detailed description, drawings, and appended claims.